This invention relates to a method of measuring bone resorption rates. More specifically, it relates to the use of peptides of the Type I collagen C-telopeptide and antibodies specific to those peptides for the measurement of specific urine and serum peptides produced during the degradation of bone collagen.
Osteoporosis is a widespread disease, afflicting an estimated 15-20 million people in the United States. The disorder is characterized by a decrease in bone mass which leads to a weakening of the bone. The degree of weakening can be severe enough to result in fractures occurring spontaneously or as a result of minor trauma. Osteoporosis is most common in postmenopausal women with 25% of the women over the age of 60 suffering from spinal compression fractures. Although these fractures are seldom fatal, the continued pain and vertebral deformity are a major source of disability in the elderly. On the other hand, hip fractures not only cause pain and disability, but also lead to death within 6 months of the acute fracture in as many as 15% of patients. That 30% of women have hip fractures by the time they reach age 90 demonstrates the magnitude of this problem. Although most common in postmenopausal women, osteoporosis can also occur in older men and younger amenorrheic women. The annual cost of care for osteoporotic-related injuries is estimated to exceed 7 billion dollars (Barnes, O. M., Science, 236:914 (1987)).
Several methods exist for determining bone mass and thus diagnosing osteoporosis. Among these are single photon absorptiometry, dual photon absorptiometry, and quantitative computed tomography. Although very good at demonstrating the present density of the bone, these methods are unable to give an accurate determination of the rate of bone loss. Because of this, present methods cannot be used to monitor the efficacy of short term therapy for the treatment of osteoporosis.
A method of determining the present rate of bone loss would be of great use in monitoring osteoporosis. Assays that are presently used for this purpose are based on the release of a specific amino acid or peptides from degraded collagen, the most common protein found in bone.
Type I collagen is unique to connective tissues and is a major component in bone. Each Type I collagen fiber is composed of three long, helical polypeptide chains (two .alpha.1 (I) chains and one .alpha.2 (I) chain) that bind tightly to each other. The normal synthesis and breakdown of this collagen type can be altered during the pathogenesis of many kinds of disease, including osteoporosis. Because bone is a metabolically active tissue throughout life, indicators of Type I collagen turnover could be useful as markers in metabolic bone disease. The major means for estimating the metabolic rate of bone collagen has been to quantify the urinary excretion of hydroxyproline which is derived from collagenous proteins. Unfortunately, this test has proven tedious, associated with several sources of error, and not specific for Type I collagen. Azria, Calcif. Tissue Int. 45:7-11 (1989). Hydroxyproline is an amino acid produced as a result of posttranslational modification of proline during collagen synthesis. It is found almost exclusively in collagen. During collagen breakdown, hydroxyproline is released into the serum and cleared from the body in urine. There are, however, some difficulties in using hydroxyproline as a marker of bone resorption. First, hydroxyproline is found in all collagens, not just type I collagen, the type found in bone. Therefore it is not a marker entirely specific for bone. Second, a large portion of the hydroxyproline produced from collagen degradation is metabolized by the liver and never appears in the urine. Finally, it has been suggested that about 10 percent of the urinary hydroxyproline is indicative of bone formation and not resorption, being released during the proteolytic processing of procollagen. Because of these problems, hydroxyproline is not a reliable quantitative method for analysis of bone resorption. Despite these drawbacks, urinary hydroxyproline remains the most commonly used marker for bone resorption.
Hydroxylysine is a second amino acid unique to collagen and collagen-like peptides. Similar to hydroxyproline, hydroxylysine is produced by a posttranslational modification of lysine during collagen synthesis. Unlike hydroxyproline, this amino acid can be further modified by glycosylation to produce galactosyl hydroxylysine (GHyl) and glucosylgalactosyl hydroxylysine (GGHyl). Although hydroxylysine is found in all collagens, the ratio of GHyl to GGHyl is higher in bone tissue than in other collagens while GGHyl is higher in skin. The urinary GGHyl:GHyl ratio in normal adults is greater than 1.0, whereas in rapidly growing children or in patients with bone disorders such as Paget's Disease, a disease characterized by increased bone resorption, the ratio of GGHyl to GHyl is less than 1.0. Unlike hydroxyproline, the hydroxylysine glycosides are not significantly metabolized prior to urinary excretion. Because of this, it has been estimated that urinary hydroxylysine glycosides can account for 50-100% of collagen breakdown, as opposed to the 10-25% estimated for hydroxyproline. Although the potential for using urinary hydroxylysine glycoside ratios as markers for bone resorption is great, the existing methods for their determination are tedious, making their measurements impractical outside of a clinical research laboratory. Because of the widespread nature of disease associated with the bone resorption, assays which cannot be performed by community clinical laboratories lack general clinical usefulness.
A third group of compounds suggested as potential markers of collagen degradation are pyridinium cross-links. Pyridinium cross-links are derived from the condensation of three hydroxylysine or lysine residues by the action of lysyl oxidase. These compounds have been shown to serve as cross-links between collagen chains and form during the maturation of collagen. Two chemical forms of pyridinium residues have been identified. Hydroxylysyl pyridinoline (HP), also referred to as pyridinoline, is found predominantly in non-bone collagens, such as cartliage. Lysyl pyridinoline (LP), also called deoxypyridinoline, is the form found predominantly in bone and dentin. During collagen breakdown, the pyridinium cross-links are released and cleared by the kidneys. Both compounds are naturally fluorescent with the same excitation and emission spectra. This allows them to be resolved and assayed directly from urine. Unfortunately, the assay involves extensive hydrolysis of urine followed by ion-exchange chromatography and reverse phase high pressure liquid chromatography. Although this method provides reproducible results, it is tedious and does not lend itself to the measurement of large numbers of clinical samples.
An enzyme-linked immunoassay to measure HP urine has been reported. (Robins, Biochem J., 207:617-620 (1982)). This assay used a rabbit polyclonal antibody raised against free HP that had been conjugated to bovine serum albumin. Using this assay, Robins was able to demonstrate differences between HP levels in urine from normal adults and patients with arthritic joint diseases. Although Robins was able to demonstrate HP levels in urine at concentrations that were proportional to the rate of joint cartilage resorption, the increase in HP could result from cartilage destruction alone and thus, reliable information on bone resorption could not be obtained from this assay.
Other immunoassays for measuring both HP and LP have been developed. Robins, Biochem. J., 207:617-620 (1982). This assay reacts only with the free crosslinks and does not react with the peptide-bound crosslinks and thus is not an accurate reflection of resorption.
Hanson and Eyre, J. Bone & Mineral Res. 6: (Suppl. 1) 251(1991) and Risteli et al., J. Bone & Mineral Res. 6: (Suppl. 1) 251(1991), have reported the development of immunoassays based on cross-linked collagen peptides found either in urine, as described by Eller, U.S. Pat. No. 4,973,666, or liberated from human femoral bone by enzyme degradation. Both these assays measure levels of pyridinoline crosslinked collagen peptides. Unfortunately, the concentration of the crosslinks in bone is not invariant. The concentration of crosslinks in bones can vary between different types of bone and furthermore, between the bone of different subjects. The pyridinoline formed in bone is a post-translational modification of the collagen molecule. This post-translational change varies with the nutritional status of the patient, with the hormonal status of the patient, with the age of the patient, and it varies considerably from patient to patient. The amount of pyridinoline appearing in the serum or urine will be a direct reflection of the amount in bone. Thus, the urine and serum concentrations of both the crosslinks themselves and the crosslinked collagen peptides depend not only on the amount of bone resorption, but also on the type of bone being resorbed and the concentration of the crosslinks in the bone. These variables limit the clinical utility of assays which measure pyridinoline or pyridinoline-crosslinked peptides because the amount of bone resorption is not predictably related to the pyridinoline measured.
What is needed is an assay measuring a degradation product of collagen, which is not influenced by diet, hormonal status, age, or disease. The method should be one that can be applied to body fluids such as serum or urine. The method also should be sensitive and able to determine rapidly bone resorption rates so that it can be used to assess various therapeutic regimens. The present invention fulfills these and other needs.